Figure 4. Isolated microparticles can be quantified and the real-time generation of neutrophil microparticles can be assessed with IS^X.

(a) Neutrophils were labeled with BODIPY-Maleimide, then microparticles were generated using TNF-α (50 ng/ml), IL-8 (50 ng/ml) or leukotriene B₄ (10 nM) and enumerated using IS^X. (b) Microparticle diameter was assessed using the Channel 02 fluorescence parameter with a 75% stringent mask applied. Data are shown as mean ± SEM of 3 blood donors. *P<0.05 and **P<0.01 using repeated measures ANOVA and Bonferroni post-test. (c) Neutrophils were isolated from three healthy donors, immediately labeled with BODIPY-Maleimide dye and stimulated with TNF-α (50 ng/ml) followed by immediate acquisition for 45 minutes. The rate of microparticle production was inferred by gating the microparticle population, applying the “Objects per second” feature and plotting against time. (d) Summarized data from the three donors, P<0.001 with Mixed-model ANOVA. (e) Representative IS^X gallery images of neutrophil microparticles budding at the times indicated.